Brassica rapa var nipposinica named origami

ABSTRACT

Novel  Brassica rapa  var.  nipposinica , such as  Brassica rapa  var.  nipposinica  designated ORIGAMI is disclosed. In some embodiments, the invention relates to the seeds of  Brassica rapa  var.  nipposinica  ORIGAMI, to the plants and plant parts of  Brassica rapa  var.  nipposinica  ORIGAMI, and to methods for producing a  Brassica rapa  var.  nipposinica  plant by crossing the  Brassica rapa  var.  nipposinica  ORIGAMI with itself or another  Brassica rapa  var.  nipposinica  plant. The invention further relates to methods for producing other  Brassica rapa  var.  nipposinica  plants derived from the  Brassica rapa  var.  nipposinica  ORIGAMI.

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims priority to, and the benefit of U.S.Provisional Patent Application Ser. No. 62/251,868, filed Nov. 6, 2015,which is hereby incorporated by reference in its entirety for allpurposes.

FIELD OF THE INVENTION

The present invention relates to the field of agriculture, to new anddistinctive Brassica rapa var. nipposinica cultivar designated ORIGAMI,and to methods of making and using such plants.

BACKGROUND OF THE INVENTION

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed inventions, or that any publication specifically orimplicitly referenced is prior art.

Brassica rapa var. nipposinica is an important and valuable vegetablecrop. Thus, a continuing goal of plant breeders is to develop stable,high yielding Brassica rapa var. nipposinica cultivars that areagronomically sound. The reasons for this goal are to maximize theamount of yield produced on the land used as well as to improve theplant agronomic qualities. To accomplish this goal, the Brassica rapavar. nipposinica breeder must select and develop Brassica rapa var.nipposinica plants that have the traits that result in superiorcultivars.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described inconjunction with systems, tools and methods which are meant to beexemplary, not limiting in scope.

In various embodiments, one or more of the above-described problems havebeen reduced or eliminated, while other embodiments are directed toother improvements.

According to the invention, in some embodiments, there is provided onenovel Brassica rapa var. nipposinica cultivar, designated ORIGAMI. Thisinvention thus relates to the seeds of Brassica rapa var. nipposinicacultivar designated ORIGAMI, to the plants or parts thereof of Brassicarapa var. nipposinica cultivar designated ORIGAMI, to plants or partsthereof consisting essentially of the phenotypic and morphologicalcharacteristics of Brassica rapa var. nipposinica cultivar designatedORIGAMI, and/or having all of the physiological and morphologicalcharacteristics of Brassica rapa var. nipposinica cultivar designatedORIGAMI and/or having one or more or all of the characteristics ofBrassica rapa var. nipposinica cultivar designated ORIGAMI listed inTable 1 including but not limited to as determined at the 5%significance level when grown in the same environmental condition,and/or having one or more of the physiological and morphologicalcharacteristics of Brassica rapa var. nipposinica cultivar designatedORIGAMI listed in Table 1 including but not limited to as determined atthe 5% significance level when grown in the same environmental conditionand/or having all of the physiological and morphological characteristicsof Brassica rapa var. nipposinica cultivar designated ORIGAMI listed inTable 1 including but not limited to as determined at the 5%significance level when grown in the same environmental conditionsand/or having all of the physiological and morphological characteristicsof Brassica rapa var. nipposinica cultivar designated ORIGAMI listed inTable 1 when grown in the same environmental conditions. The inventionalso relates to variants, mutants and trivial modifications of the seedor plant of Brassica rapa var. nipposinica cultivar designated ORIGAMI.

Plant parts of the Brassica rapa var. nipposinica cultivar of thepresent invention are also provided, such as a leaf, flower, cell,pollen or ovule obtained from the plant cultivar. The present inventionprovides leaves of the Brassica rapa var. nipposinica cultivar of thepresent invention. Such leaves could be used as fresh products forconsumption or in processes resulting in processed products such as foodproducts comprising one or more harvested part of the Brassica rapa var.nipposinica plant ORIGAMI, for example harvested leaves. The harvestedpart or food product can be or can comprise the Brassica rapa var.nipposinica leaves of the Brassica rapa var. nipposinica plant ORIGAMIor a salad mixture comprising leaves of the Brassica rapa var.nipposinica plant ORIGAMI. The food products might have undergone one ormore processing steps such as, but not limited to cutting, washing,mixing, etc. All such products are part of the present invention.

The plants and seeds of the present invention include those that may beof an essentially derived variety as defined in section 41(3) of thePlant Variety Protection Act of the United States of America, i.e., avariety that is predominantly derived from Brassica rapa var.nipposinica cultivar designated ORIGAMI or from a variety that i) ispredominantly derived from Brassica rapa var. nipposinica cultivardesignated ORIGAMI, while retaining the expression of the essentialcharacteristics that result from the genotype or combination ofgenotypes of Brassica rapa var. nipposinica cultivar designated ORIGAMI;ii) is clearly distinguishable from Brassica rapa var. nipposinicacultivar designated ORIGAMI; and iii) except for differences that resultfrom the act of derivation, conforms to the initial variety in theexpression of the essential characteristics that result from thegenotype or combination of genotypes of the initial variety or cultivar.

In another aspect, the present invention provides regenerable cells. Insome embodiments, the regenerable cells are for use in tissue culture ofBrassica rapa var. nipposinica cultivar designated ORIGAMI. In someembodiments, the tissue culture is capable of regenerating plantsconsisting essentially of the phenotypic and morphologicalcharacteristics of Brassica rapa var. nipposinica cultivar designatedORIGAMI, and/or having all of the phenotypic and morphologicalcharacteristics of Brassica rapa var. nipposinica cultivar designatedORIGAMI, and/or having the physiological and morphologicalcharacteristics of Brassica rapa var. nipposinica cultivar designatedORIGAMI, and/or having the characteristics of Brassica rapa var.nipposinica cultivar designated ORIGAMI. In one embodiment, theregenerated plants have one or more or all of the characteristics ofBrassica rapa var. nipposinica cultivar designated ORIGAMI listed inTable 1 including but not limited to as determined at the 5%significance level when grown in the same environmental conditions. Insome embodiments, the plant parts and cells used to produce such tissuecultures can be embryos, meristematic cells, seeds, callus, pollen,leaves, anthers, pistils, roots, root tips, stems, petioles, cotyledons,hypocotyls, ovaries, seed coat, fruits, endosperm, flowers, axillarybuds or the like. Protoplasts produced from such tissue culture are alsoincluded in the present invention. The Brassica rapa var. nipposinicashoots, roots and whole plants regenerated from the tissue culture, aswell as the leaves produced by said regenerated plants are also part ofthe invention. In some embodiments, the whole plants regenerated fromthe tissue culture have one, more than one, or all of the physiologicaland morphological characteristics of Brassica rapa var. nipposinicacultivar designated ORIGAMI listed in Table 1, including but not limitedto when grown in the same environmental conditions.

The invention also discloses methods for vegetatively propagating aplant of the present invention. In some embodiments, the methodscomprise collecting a part of a Brassica rapa var. nipposinica cultivardesignated ORIGAMI and regenerating a plant from said part. In someembodiments, the part can be for example a leaf cutting that is rootedinto an appropriate medium according to techniques known by the oneskilled in the art. Plants and plant parts thereof produced by suchmethods are also included in the present invention. In another aspect,the plants thereof produced by such methods consist essentially of thephenotypic and morphological characteristics of Brassica rapa var.nipposinica cultivar designated ORIGAMI, and/or having all of thephenotypic and morphological characteristics of Brassica rapa var.nipposinica cultivar designated ORIGAMI, and/or having the physiologicaland morphological characteristics of Brassica rapa var. nipposinicacultivar designated ORIGAMI, and/or having the characteristics ofBrassica rapa var. nipposinica cultivar designated ORIGAMI. In someembodiments, plants produced by such methods consist of one, more thanone, or all phenotypic and morphological characteristics of Brassicarapa var. nipposinica cultivar designated ORIGAMI listed in Table 1,including but not limited to when grown in the same environmentalconditions.

Further included in the invention are methods for producing leaves fromthe Brassica rapa var. nipposinica cultivar designated ORIGAMI. In someembodiments, the methods comprise growing a Brassica rapa var.nipposinica cultivar designated ORIGAMI to produce a Brassica rapa var.nipposinica leaf. In some embodiments, the methods further compriseharvesting the Brassica rapa var. nipposinica leaf. In some embodiments,the methods further comprise harvesting the Brassica rapa var.nipposinica leaf at an early stage of production, leading to “baby leaf”harvest. Such Brassica rapa var. nipposinica leaves, at any stage ofproduction, are part of the present invention.

Also included in this invention are methods for producing a Brassicarapa var. nipposinica plant. In some embodiments, the Brassica rapa var.nipposinica plant is produced by crossing the Brassica rapa var.nipposinica cultivar designated ORIGAMI with itself or another plant. Insome embodiments, the other plant can be a Brassica rapa var.nipposinica plant. In some embodiments, the other plant can be aBrassica rapa var. nipposinica hybrid or line. When crossed with itself,i.e. when ORIGAMI is crossed with another Brassica rapa var. nipposinicacultivar ORIGAMI or self-pollinated, Brassica rapa var. nipposinicacultivar ORIGAMI will be conserved (e.g. as an inbred). When crossedwith another, different Brassica rapa var. nipposinica plant, an F1hybrid seed is produced if the different Brassica rapa var. nipposinicaplant is an inbred and a “three-way cross” seed is produced if thedifferent Brassica rapa var. nipposinica plant is a hybrid. Such F1hybrid seed and three-way hybrid seeds and plants produced by growingsaid F1 and three-way hybrid seeds are included in the presentinvention. Methods for producing a F1 and three-way hybrid Brassica rapavar. nipposinica seed comprising crossing Brassica rapa var. nipposinicacultivar ORIGAMI plant with a different Brassica rapa var. nipposinicaline or hybrid and harvesting the resultant hybrid Brassica rapa var.nipposinica seed are also part of the invention. The hybrid Brassicarapa var. nipposinica seeds produced by the methods comprising crossingBrassica rapa var. nipposinica cultivar ORIGAMI plant with a differentBrassica rapa var. nipposinica plant and harvesting the resultant hybridBrassica rapa var. nipposinica seed are included in the invention, asare included the hybrid Brassica rapa var. nipposinica plants or partsthereof and seeds produced by said grown hybrid Brassica rapa var.nipposinica plants.

Further included in the invention are methods for producing a Brassicarapa var. nipposinica seed and plants made thereof. In some embodiments,said methods comprise self-pollinating the Brassica rapa var.nipposinica cultivar ORIGAMI and harvesting the resultant seeds.Brassica rapa var. nipposinica seeds produced by such method are alsopart of the invention.

In another embodiment, this invention also relates to methods forproducing other Brassica rapa var. nipposinica plants derived fromBrassica rapa var. nipposinica cultivar ORIGAMI and to the Brassica rapavar. nipposinica plants derived by the use of those methods.

In some embodiments, such methods for producing a Brassica rapa var.nipposinica plant derived from the Brassica rapa var. nipposinicacultivar ORIGAMI comprise (a) self-pollinating the Brassica rapa var.nipposinica cultivar ORIGAMI plant at least once to produce a progenyplant derived from Brassica rapa var. nipposinica cultivar ORIGAMI; Insome embodiment, the method further comprise (b) crossing the progenyplant derived from Brassica rapa var. nipposinica cultivar ORIGAMI withitself or a second Brassica rapa var. nipposinica plant to produce aseed of a progeny plant of a subsequent generation. In some embodiments,the methods further comprise (c) growing the progeny plant of thesubsequent generation; In some embodiments, the methods further comprise(d) crossing the progeny plant of the subsequent generation with itselfor a second Brassica rapa var. nipposinica plant to produce a Brassicarapa var. nipposinica plant further derived from the Brassica rapa var.nipposinica cultivar ORIGAMI. In further embodiments, step (b), step(c), and/or step (d) are repeated for at least 1, 2, 3, 4, 5, 6, 7, 8,9, or more generation to produce a Brassica rapa var. nipposinica plantderived from the Brassica rapa var. nipposinica cultivar ORIGAMI. Insome embodiments, within each crossing cycle, the second plant is thesame plant as the second plant in the last crossing cycle. In someembodiment, within each crossing cycle, the second plant is differentfrom the second plant of the last crossing cycle.

Another method for producing a Brassica rapa var. nipposinica plantderived from the variety ORIGAMI, comprises the steps of: (a) crossingthe ORIGAMI plant with a second Brassica rapa var. nipposinica plant toproduce a progeny plant derived from Brassica rapa var. nipposinicacultivar ORIGAMI; In some embodiments, the method further comprises (b)crossing the progeny plant derived from Brassica rapa var. nipposinicacultivar ORIGAMI with itself or a second Brassica rapa var. nipposinicaplant to produce a seed of a progeny plant of a subsequent generation.In some embodiments, the method further comprises (c) growing theprogeny plant of the subsequent generation from the seed; In someembodiments, the method further comprises (d) crossing the progeny plantof the subsequent generation with itself or a different Brassica rapavar. nipposinica plant to produce a Brassica rapa var. nipposinica plantderived from ORIGAMI. In a further embodiment, step (b), step (c),and/or step (d) are repeated for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, ormore generation to produce a Brassica rapa var. nipposinica plantderived from ORIGAMI. In some embodiments, within each crossing cycle,the second plant is the same plant as the second plant in the lastcrossing cycle. In some embodiments, within each crossing cycle, thesecond plant is different from the second plant in the last crossingcycle.

In another aspect, the present invention provides methods of introducingor modifying one or more desired trait(s) into the Brassica rapa var.nipposinica cultivar ORIGAMI and plants or seeds obtained from suchmethods. The desired trait(s) may be, but not exclusively, a singlegene. In some embodiments, the gene is a dominant allele. In someembodiments, the gene is a partially dominant allele. In someembodiments, the gene is a recessive allele. In some embodiments, thegene or genes will confer such traits as male sterility, herbicideresistance, insect resistance, resistance for bacterial, fungal,mycoplasma or viral disease, improved shelf life, water-stresstolerance, delayed senescence or controlled ripening, enhanced plantquality such as improved drought or salt tolerance, enhanced plantvigor, improved or changed colors or improved fresh cut application. Forthe present invention and the skilled artisan, disease is understood toinclude, but not limited to fungal diseases, viral diseases, bacterialdiseases, mycoplasma diseases, or other plant pathogenic diseases and adisease resistant plant will encompass a plant resistant to fungal,viral, bacterial, mycoplasma, and other plant pathogens The gene orgenes may be naturally occurring Brassica rapa var. nipposinica gene(s),mutant(s) or genes modified through New Breeding Techniques. In someembodiments, the method for introducing the desired trait(s) is abackcrossing process making use of a series of backcrosses to Brassicarapa var. nipposinica cultivar ORIGAMI during which the desired trait(s)is maintained by selection. The single gene conversion plants that canbe obtained by the method are included in the present invention.

When dealing with a gene that has been modified, for example through NewBreeding Techniques, the trait (genetic modification) could be directlymodified into the newly developed line/cultivar such as Brassica rapavar. nipposinica cultivar ORIGAMI. Alternatively, if the trait is notmodified into each newly developed line/cultivar such as Brassica rapavar. nipposinica cultivar ORIGAMI, another typical method used bybreeders of ordinary skill in the art to incorporate the modified geneis to take a line already carrying the gene and to use such line as adonor line to transfer the gene into the newly developed line. The samewould apply for a naturally occurring trait or one arising fromspontaneous or induced mutations.

In some embodiments, the backcross breeding process of Brassica rapavar. nipposinica cultivar ORIGAMI comprises (a) crossing Brassica rapavar. nipposinica cultivar ORIGAMI with plants that comprise the desiredtrait(s) to produce F1 progeny plants. In some embodiments, the processfurther comprises (b) selecting the F1 progeny plants that have thedesired trait(s); In some embodiments, the process further comprises (c)crossing the selected progeny plants with the Brassica rapa var.nipposinica cultivar ORIGAMI plants to produce backcross progeny plants;In some embodiments, the process further comprises (d) selecting forbackcross progeny plants that have the desired trait(s) andphysiological and morphological characteristics of the Brassica rapavar. nipposinica cultivar ORIGAMI to produce selected backcross progenyplants; In some embodiments, the process further comprises (e) repeatingsteps (c) and (d) one, two, three, four, five six, seven, eight, nine ormore times in succession to produce selected, second, third, fourth,fifth, sixth, seventh, eighth, ninth or higher backcross progeny plantsthat have the desired trait(s) and consist essentially of the phenotypicand morphological characteristics of the Brassica rapa var. nipposinicacultivar ORIGAMI, and/or have all of the phenotypic and morphologicalcharacteristics of the Brassica rapa var. nipposinica cultivar ORIGAMI,and/or have the desired trait(s) and the physiological and morphologicalcharacteristics of the Brassica rapa var. nipposinica cultivar ORIGAMIas determined in Table 1, including but not limited to when grown in thesame environmental conditions or including but not limited to at a 5%significance level when grown in the same environmental conditions. Insome embodiments, the backcross breeding process of Brassica rapa var.nipposinica cultivar of ORIGAMI comprises the following steps: (a)crossing Brassica rapa var. nipposinica cultivar ORIGAMI with plants ofanother line that comprise the desired trait(s) to produce F1 progenyplants, (b) selecting the F1 progeny plants that have the desiredtrait(s); (c) crossing the selected progeny plants with the Brassicarapa var. nipposinica cultivar ORIGAMI plants to produce backcrossprogeny plants; (d) selecting for backcross progeny plants that have thedesired trait(s) and physiological and morphological characteristics ofthe Brassica rapa var. nipposinica cultivar ORIGAMI to produce selectedbackcross progeny plants; and (e) repeating steps (c) and (d) one, two,three, four, five six, seven, eight, nine or more times in succession toproduce selected, second, third, fourth, fifth, sixth, seventh, eighth,ninth or higher backcross progeny plants that consist essentially of thephenotypic and morphological characteristics of the Brassica rapa var.nipposinica cultivar ORIGAMI, and/or have all of the phenotypic andmorphological characteristics of the Brassica rapa var. nipposinicacultivar ORIGAMI, and/or have the desired trait and the physiologicaland morphological characteristics of the Brassica rapa var. nipposinicacultivar ORIGAMI as determined in Table 1, including but not limited toat a 5% significance level when grown in the same environmentalconditions. The Brassica rapa var. nipposinica plants or seeds producedby the methods are also part of the invention. Backcrossing breedingmethods, well known to one skilled in the art of plant breeding will befurther developed in subsequent parts of the specification.

In an embodiment of this invention is a method of making a backcrossconversion of Brassica rapa var. nipposinica cultivar ORIGAMI. In someembodiments, the method comprises crossing Brassica rapa var.nipposinica cultivar ORIGAMI with a donor plant comprising a mutantgene(s), a naturally occurring gene(s) or a gene(s) and/or nucleotidesequences modified through the use of New Breeding Techniques conferringone or more desired trait to produce F1 progeny plant. In someembodiment, the method further comprises selecting the F1 progeny plantcomprising the naturally occurring gene(s), mutant gene(s) or modifiedgene(s) and/or nucleotide sequences conferring the one or more desiredtrait. In some embodiments, the method further comprises backcrossingthe selected progeny plant to the Brassica rapa var. nipposinicacultivar ORIGAMI. This method may further comprise the step of obtaininga molecular marker profile of the Brassica rapa var. nipposinicacultivar ORIGAMI and using the molecular marker profile to select forthe progeny plant with the desired trait and the molecular markerprofile of the Brassica rapa var. nipposinica cultivar ORIGAMI. Theplants or parts thereof produced by such methods are also part of thepresent invention.

In some embodiments of the invention, the number of loci that may bebackcrossed into the Brassica rapa var. nipposinica cultivar ORIGAMI isat least 1, 2, 3, 4, 5, 6, 7, 8, 9, or more. A single locus may containone or several genes. A single locus conversion also allows for makingone or more site specific changes to the plant genome, such as, withoutlimitation, one or more nucleotide change, deletion, insertions, etc. Insome embodiments, the single locus conversion is performed by genomeediting, a.k.a. genome editing with engineered nucleases (GEEN). In someembodiments, the genome editing comprises using one or more engineerednucleases. In some embodiments, the engineered nucleases include, butare not limited to Zinc finger nucleases (ZFNs), TranscriptionActivator-Like Effector Nucleases (TALENs), the CRISPR/Cas system,engineered meganuclease re-engineered homing endonucleases, andendonucleases for DNA guided genome editing (Gao et al., NatureBiotechnology (2016), doi: 10.1038/nbt.3547). In some embodiments, thesingle locus conversion changes one or several nucleotides of the plantgenome. Such genome editing techniques are some of the techniques nowknown by a person skilled in the art and herein are collectivelyreferred to as ‘New Breeding Techniques’.

The invention further provides methods for developing Brassica rapa var.nipposinica plants in a Brassica rapa var. nipposinica plant breedingprogram using plant breeding techniques including but not limited to,recurrent selection, backcrossing, pedigree breeding, molecular marker(Isozyme Electrophoresis, Restriction Fragment Length Polymorphisms(RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily PrimedPolymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting(DAF), Sequence Characterized Amplified Regions (SCARs), AmplifiedFragment Length Polymorphisms (AFLPs), and Simple Sequence Repeats(SSRs) which are also referred to as Microsatellites, Single NucleotidePolymorphisms (SNPs), etc.) enhanced selection, genetic marker enhancedselection and transformation. Seeds, Brassica rapa var. nipposinicaplants, and parts thereof produced by such breeding methods are alsopart of the invention.

The invention also relates to variants, mutants and trivialmodifications of the seed or plant of the Brassica rapa var. nipposinicacultivar ORIGAMI. Variants, mutants and trivial modifications of theseed or plant of Brassica rapa var. nipposinica cultivar ORIGAMI can begenerated by methods available to one skilled in the art, including butnot limited to, mutagenesis (e.g., chemical mutagenesis, radiationmutagenesis, transposon mutagenesis, insertional mutagenesis, signaturetagged mutagenesis, site-directed mutagenesis, and natural mutagenesis),knock-outs/knock-ins, antisense, RNA interference and other techniquessuch as the New Breeding Techniques. For more information of mutagenesisin plants, such as agents, protocols, see Acquaah et al. (Principles ofplant genetics and breeding, Wiley-Blackwell, 2007, ISBN 1405136464,9781405136464, which is herein incorporated by reference in its entity).

The invention also relates to a mutagenized population of the Brassicarapa var. nipposinica cultivar ORIGAMI and methods of using suchpopulations. In some embodiments, the mutagenized population can be usedin screening for new Brassica rapa var. nipposinica plants whichcomprises one or more or all of the morphological and physiologicalcharacteristics of Brassica rapa var. nipposinica cultivar ORIGAMI. Insome embodiments, the new Brassica rapa var. nipposinica plants obtainedfrom the screening process comprise all of the morphological andphysiological characteristics of the Brassica rapa var. nipposinicacultivar ORIGAMI, and one or more additional or different morphologicaland physiological characteristics that Brassica rapa var. nipposinicacultivar ORIGAMI does not have.

This invention also is directed to methods for producing a Brassica rapavar. nipposinica plant by crossing a first parent Brassica rapa var.nipposinica plant with a second parent Brassica rapa var. nipposinicaplant wherein either the first or second parent Brassica rapa var.nipposinica plant is a Brassica rapa var. nipposinica cultivar ORIGAMI.Further, both first and second parent Brassica rapa var. nipposinicaplants can come from the Brassica rapa var. nipposinica cultivarORIGAMI. Further, the Brassica rapa var. nipposinica cultivar ORIGAMIcan be self-pollinated i.e. the pollen of a Brassica rapa var.nipposinica cultivar ORIGAMI can pollinate the ovule of the sameBrassica rapa var. nipposinica cultivar ORIGAMI, respectively. Whencrossed with another Brassica rapa var. nipposinica plant, a hybrid seedis produced. Such methods of hybridization and self-pollination are wellknown to those skilled in the art of breeding.

A Brassica rapa var. nipposinica cultivar such as Brassica rapa var.nipposinica cultivar ORIGAMI has been produced through several cycles ofself-pollination and is therefore to be considered as a homozygous plantor line. An inbred line can also be produced through the dihaploidsystem which involves doubling the chromosomes from a haploid plant orembryo thus resulting in an inbred line that is genetically stable(homozygous) and can be reproduced without altering the inbred line:Haploid plants could be obtained from haploid embryos that might beproduced from microspores, pollen, anther cultures or ovary cultures orspontaneous haploidy. The haploid embryos may then be doubled bychemical treatments such as by colchicine or be doubled autonomously.The haploid embryos may also be grown into haploid plants and treated toinduce the chromosome doubling. In either case, fertile homozygousplants are obtained. A hybrid variety is classically created through thefertilization of an ovule from an inbred parental line by the pollen ofanother, different inbred parental line. Due to the homozygous state ofthe inbred line, the produced gametes carry a copy of each parentalchromosome. As both the ovule and the pollen bring a copy of thearrangement and organization of the genes present in the parental lines,the genome of each parental line is present in the resulting F1 hybrid,theoretically in the arrangement and organization created by the plantbreeder in the original parental line.

As long as the homozygosity of the parental lines is maintained, theresulting hybrid cross shall be stable. The F1 hybrid is then acombination of phenotypic characteristics issued from two arrangementand organization of genes, both created by a man skilled in the artthrough the breeding process.

Still further, this invention also is directed to methods for producinga Brassica rapa var. nipposinica cultivar ORIGAMI-derived Brassica rapavar. nipposinica plant by crossing Brassica rapa var. nipposinicacultivar ORIGAMI with a second Brassica rapa var. nipposinica plant. Insome embodiments, the method further comprises obtaining a progeny seedfrom the cross. In some embodiment, the method further comprises growingthe progeny seed, and possibly repeating the crossing and growing stepswith the Brassica rapa var. nipposinica cultivar ORIGAMI-derived plantfrom 0 to 7, or more times. Thus, any such methods using the Brassicarapa var. nipposinica cultivar ORIGAMI are part of this invention:selfing, backcrosses, hybrid production, crosses to populations, and thelike. All plants produced using Brassica rapa var. nipposinica cultivarORIGAMI as a parent are within the scope of this invention, includingplants derived from Brassica rapa var. nipposinica cultivar ORIGAMI. Insome embodiment, such plants have one, more than one, or all phenotypicand morphological characteristics of Brassica rapa var. nipposinicacultivar ORIGAMI listed in Table 1 including but not limited to whengrown in the same environmental conditions.

Such plants might exhibit additional and desired characteristics ortraits such as high seed yield, high seed germination, seedling vigor,early maturity, high yield, disease tolerance or resistance, andadaptability for soil and climate conditions. Consumer-driven traits,such as a preference for a given leaf size, shape, color, texture,taste, are other traits that may be incorporated into new Brassica rapavar. nipposinica plants developed by this invention.

A Brassica rapa var. nipposinica plant can also be propagatedvegetatively. A part of the plant, for example a shoot tissue, iscollected, and a new plant is obtained from the part. Such parttypically comprises an apical meristem of the plant. The collected partis transferred to a medium allowing development of a plantlet, includingfor example rooting or development of shoots. This is achieved usingmethods well-known in the art. Accordingly, in one embodiment, a methodof vegetatively propagating a plant of the present invention comprisescollecting a part of a plant according to the present invention, e.g. ashoot tissue, and obtaining a plantlet from said part. In oneembodiment, a method of vegetatively propagating a plant of the presentinvention comprises: a) collecting tissue of a plant of the presentinvention; b) rooting said proliferated shoots to obtain rootedplantlets. In one embodiment, a method of vegetatively propagating aplant of the present invention comprises: a) collecting tissue of aplant of the present invention; b) cultivating said tissue to obtainproliferated shoots; c) rooting said proliferated shoots to obtainrooted plantlets. In one embodiment, such method further comprisesgrowing a plant from said plantlets. In one embodiment, leaves areharvested from said plant. In one embodiment, the leaves are processedinto products prepared cut leaves.

In some embodiments, the present invention teaches a seed of Brassicarapa var. nipposinica cultivar ORIGAMI, wherein a representative sampleof seed of said Brassica rapa var. nipposinica cultivar is depositedunder NCIMB No ______.

In some embodiments, the present invention teaches a Brassica rapa var.nipposinica plant, or a part thereof, produced by growing the depositedORIGAMI seed.

In some embodiments, the present invention teaches Brassica rapa var.nipposinica plant parts, wherein the Brassica rapa var. nipposinica partis selected from the group consisting of: a leaf, a flower, an ovule,pollen, and a cell.

In some embodiments, the present invention teaches a Brassica rapa var.nipposinica plant, or a part thereof, having all of the characteristicsof Brassica rapa var. nipposinica cultivar ORIGAMI as listed in Table 1of this application including but not limited to when grown in the sameenvironmental conditions.

In some embodiments, the present invention teaches a Brassica rapa var.nipposinica plant, or a part thereof, having all of the physiologicaland morphological characteristics of Brassica rapa var. nipposinicacultivar ORIGAMI, wherein a representative sample of seed of saidBrassica rapa var. nipposinica plant was deposited under NCIMB No______.

In some embodiments, the present invention teaches a tissue culture ofregenerable cells produced from the plant or plant part grown from thedeposited Brassica rapa var. nipposinica cultivar ORIGAMI seed, whereincells of the tissue culture are produced from a plant part selected fromthe group consisting of protoplasts, embryos, meristematic cells,callus, pollen, ovules, flowers, seeds, leaves, roots, root tips,anthers, stems, petioles, head, axillary buds, cotyledons andhypocotyls. In some embodiments, the plant part includes protoplastsproduced from a plant grown from the deposited Brassica rapa var.nipposinica cultivar ORIGAMI seed.

In some embodiments, the present invention teaches a Brassica rapa var.nipposinica plant regenerated from the tissue culture from a plant grownfrom the deposited Brassica rapa var. nipposinica cultivar ORIGAMI seed,said plant having the characteristics of Brassica rapa var. nipposinicacultivar ORIGAMI, wherein a representative sample of seed of saidBrassica rapa var. nipposinica cultivar ORIGAMI is deposited under NCIMBNo ______.

In some embodiments, the present invention teaches a Brassica rapa var.nipposinica leaf produced from plants grown from the deposited Brassicarapa var. nipposinica cultivar ORIGAMI seed.

In some embodiments, the methods of producing said Brassica rapa var.nipposinica leaf comprise a) growing the Brassica rapa var. nipposinicaplant from deposited Brassica rapa var. nipposinica cultivar ORIGAMIseed to produce a Brassica rapa var. nipposinica leaf, and b) harvestingsaid Brassica rapa var. nipposinica leaf. In some embodiments, thepresent invention also teaches a Brassica rapa var. nipposinica leafproduced by the method of producing Brassica rapa var. nipposinica leafas described above.

In some embodiments, the present invention teaches methods for producinga Brassica rapa var. nipposinica seed comprising crossing a first parentBrassica rapa var. nipposinica plant with a second parent Brassica rapavar. nipposinica plant and harvesting the resultant Brassica rapa var.nipposinica seed, wherein said first parent Brassica rapa var.nipposinica plant and/or second parent Brassica rapa var. nipposinicaplant is the Brassica rapa var. nipposinica plant produced from thedeposited Brassica rapa var. nipposinica cultivar ORIGAMI seed, or aBrassica rapa var. nipposinica plant having all of the characteristicsof Brassica rapa var. nipposinica cultivar ORIGAMI as listed in Table 1of this application including but not limited to when grown in the sameenvironmental conditions.

In some embodiments, the present invention teaches methods for producinga Brassica rapa var. nipposinica seed comprising self-pollinating theBrassica rapa var. nipposinica plant grown from the deposited Brassicarapa var. nipposinica cultivar ORIGAMI seed and harvesting the resultantBrassica rapa var. nipposinica seed.

In some embodiments, the present invention teaches the seed produced byany of the above described methods.

In some embodiments, the present invention teaches methods ofvegetatively propagating the Brassica rapa var. nipposinica plant grownfrom the deposited Brassica rapa var. nipposinica cultivar ORIGAMI seed,said method comprising a) collecting part of a plant grown from thedeposited Brassica rapa var. nipposinica cultivar ORIGAMI seed and b)regenerating a plant from said part.

In some embodiments, the method further comprises harvesting a leaf fromsaid vegetatively propagated plant.

In some embodiments, the present invention teaches the plant, the leavesthereof of plants vegetatively propagated from plant parts of plantsgrown from the deposited Brassica rapa var. nipposinica cultivar ORIGAMIseed.

In some embodiments, the present invention teaches methods of producinga Brassica rapa var. nipposinica plant derived from the Brassica rapavar. nipposinica cultivar ORIGAMI.

In some embodiments, the methods comprise (a) self-pollinating the plantgrown from the deposited Brassica rapa var. nipposinica cultivar ORIGAMIseed at least once to produce a progeny plant derived from Brassica rapavar. nipposinica cultivar ORIGAMI. In some embodiment, the methodfurther comprises (b) crossing the progeny plant derived from Brassicarapa var. nipposinica cultivar ORIGAMI with itself or a second Brassicarapa var. nipposinica plant to produce a seed of a progeny plant of asubsequent generation; and (c) growing the progeny plant of thesubsequent generation from the seed, and crossing the progeny plant ofthe subsequent generation with itself or a second Brassica rapa var.nipposinica plant to produce a Brassica rapa var. nipposinica plantderived from the Brassica rapa var. nipposinica cultivar ORIGAMI. Insome embodiments said method further comprises the step of: (d)repeating steps (b) and/or (c) for at least 1, 2, 3, 4, 5, 6, 7, 8, 9,or more generation to produce a Brassica rapa var. nipposinica plantderived from the Brassica rapa var. nipposinica cultivar ORIGAMI.

In some embodiments, the present invention teaches methods of producinga Brassica rapa var. nipposinica plant derived from the Brassica rapavar. nipposinica cultivar ORIGAMI, the methods comprising (a) crossingthe plant grown from the deposited Brassica rapa var. nipposinicacultivar ORIGAMI seed with a second Brassica rapa var. nipposinica plantto produce a progeny plant derived from Brassica rapa var. nipposinicacultivar ORIGAMI. In some embodiments, the method further comprises (b)crossing the progeny plant derived from Brassica rapa var. nipposinicacultivar ORIGAMI with itself or a second Brassica rapa var. nipposinicaplant to produce a seed of a progeny plant of a subsequent generationand, (c) growing the progeny plant of the subsequent generation from theseedand crossing the progeny plant of the subsequent generation withitself or a second Brassica rapa var. nipposinica plant to produce aBrassica rapa var. nipposinica plant derived from the Brassica rapa var.nipposinica cultivar ORIGAMI. In some embodiments said method furthercomprises the step of (d) repeating steps (b), and/or (c) for at least1, 2, 3, 4, 5, 6, 7, 8, 9, or more generation to produce a Brassica rapavar. nipposinica plant derived from the Brassica rapa var. nipposinicacultivar ORIGAMI.

In some embodiments, the present invention teaches plants grown from thedeposited Brassica rapa var. nipposinica cultivar ORIGAMI seed whereinsaid plants comprise at least one single locus conversion. In someembodiments said single locus conversion confers said plant with a traitselected from the group consisting of male sterility, male fertility,herbicide resistance, insect resistance, disease resistance, waterstress tolerance, heat tolerance, delayed senescence, improved ripeningcontrol, long shelf life, and improved salt tolerance when compared to asuitable check plant. In some embodiments, the check plant is a Brassicarapa var. nipposinica cultivar ORIGAMI plant not having said singlelocus conversion. In some embodiments, the at least one single locusconversion is an artificially mutated gene or a gene or nucleotidesequence modified through the use of New Breeding Techniques.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by study of thefollowing descriptions.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In the description and tables that follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Allele. An allele is any of one or more alternative forms of a genewhich relate to one trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotype of the F₁ hybrid.

Essentially all of the physiological and morphological characteristics.A plant having essentially all of the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics of the recurrent parent, except for the characteristicsderived from the converted gene.

First water date. The date the seed first receives adequate moisture togerminate. This can and often does equal the planting date

Immunity to disease(s) and or insect(s). A Brassica rapa var.nipposinica plant which is not subject to attack or infection byspecific disease(s) and or insect(s) is considered immune.

Intermediate resistance to disease(s) and or insect(s). A Brassica rapavar. nipposinica plant that restricts the growth and development ofspecific disease(s) and or insect(s), but may exhibit a greater range ofsymptoms or damage compared to resistant plants. Intermediate resistantplants will usually show less severe symptoms or damage than susceptibleplant varieties when grown under similar environmental conditions and/orspecific disease(s) and or insect(s) pressure, but may have heavy damageunder heavy pressure. Intermediate resistant Brassica rapa var.nipposinica plants are not immune to the disease(s) and or insect(s).

Maturity (Date). Maturity refers to the stage when plants are of fullsize or optimum weight, and in marketable form or shape to be ofcommercial or economic value. In lettuce leaf types they range from50-75 days from time of seeding, depending upon the season of the year.In lettuce baby leaf type, they range from 30 to 45 days after planting.In Brassica rapa var. nipposinica types, they range from 25 to 40 daysfrom time of seeding.

New Breeding Techniques: New breeding techniques are said of various newtechnologies developed and/or used to create new characteristics inplants through genetic variation, the aim being targeted mutagenesis,targeted introduction of new genes or gene silencing (RdDM). Examples ofsuch new breeding techniques are targeted sequence changes facilitatedthru the use of Zinc finger nuclease (ZFN) technology (ZFN-1, ZFN-2 andZFN-3, see U.S. Pat. No. 9,145,565, incorporated by reference in itsentirety), Oligonucleotide directed mutagenesis (ODM), Cisgenesis andintragenesis, RNA-dependent DNA methylation (RdDM, which does notnecessarily change nucleotide sequence but can change the biologicalactivity of the sequence), Grafting (on GM rootstock), Reverse breeding,Agro-infiltration (agro-infiltration “sensu stricto”, agro-inoculation,floral dip), Transcription Activator-Like Effector Nucleases (TALENs,see U.S. Pat. Nos. 8,586,363 and 9,181,535, incorporated by reference intheir entireties), the CRISPR/Cas system (see U.S. Pat. Nos. 8,697,359;8,771,945; 8,795,965; 8,865,406; 8,871,445; 8,889,356; 8,895,308;8,906,616; 8,932,814; 8,945,839; 8,993,233; and 8,999,641, which are allhereby incorporated by reference), engineered meganuclease re-engineeredhoming endonucleases, DNA guided genome editing (Gao et al., NatureBiotechnology (2016), doi: 10.1038/nbt.3547, incorporated by referencein its entirety), and Synthetic genomics. A complete description of eachof these techniques can be found in the report made by the JointResearch Center (JRC) Institute for Prospective Technological Studies ofthe European Commission in 2011 and titled “New plant breedingtechniques—State-of-the-art and prospects for commercial development”,which is incorporated by reference in its entirety.

Plant adaptability. A plant having good plant adaptability means a plantthat will perform well in different growing conditions and seasons.

Plant Cell, As used herein, the term plant cell includes plant cellswhether isolated, in tissue culture or incorporated in a plant or plantpart.

Plant Part. As used herein, the term plant part includes plant cells,plant protoplasts, plant cell tissue cultures from which Brassica rapavar. nipposinica plants can be regenerated, plant calli, plant clumpsand plant cells that are intact in plants or parts of plants, such asembryos, pollen, ovules, flowers, seeds, rootstock, scions, stems,roots, anthers, pistils, root tips, leaves, meristematic cells, axillarybuds, hypocotyls cotyledons, ovaries, seed coat endosperm and the like.

Quantitative Trait Loci (QTL) Quantitative trait loci refer to geneticloci that control to some degree numerically representable traits thatare usually continuously distributed.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

Resistance to disease(s) and or insect(s). A Brassica rapa var.nipposinica plant that restricts highly the growth and development ofspecific disease(s) and or insect(s) under normal disease(s) and orinsect(s) attack pressure when compared to susceptible plants. TheseBrassica rapa var. nipposinica plants can exhibit some symptoms ordamage under heavy disease(s) and or insect(s) pressure. ResistantBrassica rapa var. nipposinica plants are not immune to the disease(s)and or insect(s).

RHS. RHS refers to the Royal Horticultural Society of England whichpublishes an official botanical color chart quantitatively identifyingcolors according to a defined numbering system. The chart may bepurchased from Royal Hort. Society Enterprise Ltd. RHS Garden; Wisley,Woking, Surrey GU236QB, UK.

Single gene converted (conversion). Single gene converted (conversion)plants refer to plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of a plant are recovered in additionto the single gene transferred into the plant via the backcrossingtechnique or via genetic engineering. A single gene converted plant canalso be referred to a plant obtained though mutagenesis or through theuse of some new breeding techniques, whereas the single gene convertedplant has essentially all of the desired morphological and physiologicalcharacteristics of the original variety in addition to the single geneor nucleotide sequence muted or engineered through the new breedingtechniques.

Susceptible to disease(s) and or insect(s). A Brassica rapa var.nipposinica plant that is susceptible to disease(s) and or insect(s) isdefined as a Brassica rapa var. nipposinica plant that has the inabilityto restrict the growth and development of specific disease(s) and orinsect(s). Plants that are susceptible will show damage when infectedand are more likely to have heavy damage under moderate levels ofspecific disease(s) and or insect(s).

Tolerance to abiotic stresses. A Brassica rapa var. nipposinica plantthat is tolerant to abiotic stresses has the ability to endure abioticstress without serious consequences for growth, appearance and yield.

Uniformity. Uniformity, as used herein, describes the similarity betweenplants or plant characteristics which can be a described by qualitativeor quantitative measurements.

Variety. A plant variety as used by one skilled in the art of plantbreeding means a plant grouping within a single botanical taxon of thelowest known rank which can be defined by the expression of thecharacteristics resulting from a given genotype or combination ofphenotypes, distinguished from any other plant grouping by theexpression of at least one of the said characteristics and considered asa unit with regard to its suitability for being propagated unchanged(International convention for the protection of new varieties ofplants).

Brassica rapa var. nipposinica Plants

Brassica rapa var. nipposinica is an oriental vegetable which has beengrown for many years in Japan and which is becoming increasingly popularin the USA with the recent development of the industrial culture ofready-to-eat fresh products, in salad mix, dressing and garnishing ingourmet cuisines. This new trend has lead to the development ofbaby-leaf products such as the ones from ORIGAMI. Delicate leaves arevery attractive and appetizing, excellent for dressing gourmet food.They are grown at high concentration and harvested at very young or“baby leaf” stage, typically 25 to 40 days after planting. The plantingare often done on wider 80 to 84 inch beds and often containing up toone million plants per acre.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possesses the traits tomeet the program goals. The goal is to combine in a single variety orhybrid an improved combination of desirable traits from the parentalgermplasm.

In Brassica rapa var. nipposinica, these important traits may includeheavier texture, improved uniformity, especially of the leaves, stem andhypocotyl, higher seed yield, improved color, resistance to diseases andinsects, tolerance to drought and heat, better post-harvest shelf-lifeof the leaves, better standing ability in the field, and betteragronomic quality.

In some embodiments, particularly desirable traits that may beincorporated by this invention are improved resistance to differentviral, fungal, and bacterial pathogens. Important diseases include butare not limited to fungi such as Bremia lactucae, Fusarium oxysporum,Sclerotinia minor or sclerotorum, Botrytis cinerea, Rhizictonia solani,Microdochium panattonianum, Verticiulium dahliae, Erysiphe chicocearumor Pithium tracheiphilum, virus, such as LMV (Brassica rapa var.nipposinica mosaic virus), TSWV (tomato potted wilt virus), “Big vein”(composed of LBVV (Brassica rapa var. nipposinica big vein virus) andMILV (miratiori Brassica rapa var. nipposinica virus)), TBSV (tomatobushy stunt virus), LNSV (Brassica rapa var. nipposinica necrotic stuntvirus), TuMV (turnip mosaic virus), CMV (cucumber mosaic virus) or BWYV(beet western yellows virus), bacteria such as Pseudomonas, Xanthomonasor Rhizomonas. Improved resistance to insect pests is another desirabletrait that may be incorporated into new Brassica rapa var. nipposinicaplants developed by this invention. Insect pests affecting the variousspecies of Brassica rapa var. nipposinica include Nasonovia ribisnigri,Myzus persicae, Macrosiphum euphorbia, Nematodes pratylenchus ormeloidogyne, leafminers: Liriomyza huidobrensis or Pemphigus busarius.

Brassica rapa var. nipposinica Breeding

The goal of Brassica rapa var. nipposinica breeding is to develop new,unique and superior Brassica rapa var. nipposinica cultivar and hybrids.The breeder initially selects and crosses two or more parental lines,followed by repeated selfing and selection, producing many new geneticcombinations. Another method used to develop new, unique and superiorBrassica rapa var. nipposinica cultivar occurs when the breeder selectsand crosses two or more parental lines followed by haploid induction andchromosome doubling that result in the development of dihaploidcultivars. The breeder can theoretically generate billions of differentgenetic combinations via crossing, selfing and mutations and the same istrue for the utilization of the dihaploid breeding method.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made, during and at the end of the growing season. The cultivarsdeveloped are unpredictable. This unpredictability is because thebreeder's selection occurs in unique environments, with no control atthe DNA level (using conventional breeding procedures or dihaploidbreeding procedures), and with millions of different possible geneticcombinations being generated. A breeder of ordinary skill in the artcannot predict the final resulting cultivars he develops, exceptpossibly in a very gross and general fashion. This unpredictabilityresults in the expenditure of large research monies to develop superiornew Brassica rapa var. nipposinica cultivars.

The development of commercial Brassica rapa var. nipposinica cultivarrequires the development and selection of Brassica rapa var. nipposinicaplants, the crossing of these plants, and the evaluation of the crosses.

Pedigree breeding and recurrent selection breeding methods are used todevelop cultivars from breeding populations. Breeding programs combinedesirable traits from two or more cultivars or various broad-basedsources into breeding pools from which cultivars are developed byselfing and selection of desired phenotypes or through the dihaploidbreeding method followed by the selection of desired phenotypes. The newcultivars are evaluated to determine which have commercial potential.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, recurrent selection, andbackcross breeding.

i Pedigree Selection

Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents possessing favorable, complementary traits are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁s or by intercrossing two F₁s (sib mating). The dihaploid breedingmethod could also be used. Selection of the best individuals is usuallybegun in the F₂ population; then, beginning in the F₃, the bestindividuals in the best families are selected. Replicated testing offamilies, or hybrid combinations involving individuals of thesefamilies, often follows in the F₄ generation to improve theeffectiveness of selection for traits with low heritability. At anadvanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease of new cultivars. Similarly, the development of new cultivarsthrough the dihaploid system requires the selection of the cultivarsfollowed by two to five years of testing in replicated plots.

ii Backcross Breeding

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line which is the recurrent parent. The source of the trait tobe transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parentare selected and repeatedly crossed (backcrossed) to the recurrentparent. The resulting plant is expected to have the attributes of therecurrent parent (e.g., cultivar) and the desirable trait transferredfrom the donor parent.

When the term Brassica rapa var. nipposinica cultivar is used in thecontext of the present invention, this also includes any Brassica rapavar. nipposinica cultivar plant where one or more desired trait has beenintroduced through backcrossing methods, whether such trait is anaturally occurring one, a mutant or a transgenic one. Backcrossingmethods can be used with the present invention to improve or introduceone or more characteristic into the Brassica rapa var. nipposinicacultivar of the present invention. The term “backcrossing” as usedherein refers to the repeated crossing of a hybrid progeny back to therecurrent parent, i.e., backcrossing one, two, three, four, five, six,seven, eight, nine, or more times to the recurrent parent. The parentalBrassica rapa var. nipposinica cultivar plant which contributes the geneor the genes for the desired characteristic is termed the nonrecurrentor donor parent. This terminology refers to the fact that thenonrecurrent parent is used one time in the backcross protocol andtherefore does not recur. The parental Brassica rapa var. nipposinicacultivar to which the gene or genes from the nonrecurrent parent aretransferred is known as the recurrent parent as it is used for severalrounds in the backcrossing protocol.

In a typical backcross protocol, the original cultivar of interest(recurrent parent) is crossed to a second cultivar (nonrecurrent parent)that carries the gene or genes of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a Brassica rapa var.nipposinica plant is obtained wherein all of the desired morphologicaland physiological characteristics of the recurrent parent are recoveredin the converted plant, generally determined at a 5% significance levelwhen grown in the same environmental conditions, in addition to the geneor genes transferred from the nonrecurrent parent. It has to be notedthat some, one, two, three or more, self-pollination and growing ofpopulation might be included between two successive backcrosses. Indeed,an appropriate selection in the population produced by theself-pollination, i.e. selection for the desired trait and physiologicaland morphological characteristics of the recurrent parent might beequivalent to one, two or even three additional backcrosses in acontinuous series without rigorous selection, saving then time, moneyand effort to the breeder. A non-limiting example of such a protocolwould be the following: a) the first generation F1 produced by the crossof the recurrent parent A by the donor parent B is backcrossed to parentA, b) selection is practiced for the plants having the desired trait ofparent B, c) selected plant are self-pollinated to produce a populationof plants where selection is practiced for the plants having the desiredtrait of parent B and physiological and morphological characteristics ofparent A, d) the selected plants are backcrossed one, two, three, four,five, six, seven, eight, nine, or more times to parent A to produceselected backcross progeny plants comprising the desired trait of parentB and the physiological and morphological characteristics of parent A.Step (c) may or may not be repeated and included between the backcrossesof step (d).

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute one or more trait(s) or characteristic(s) in theoriginal inbred parental line in order to find it then in the hybridmade thereof. To accomplish this, a gene or genes of the recurrentinbred is modified or substituted with the desired gene or genes fromthe nonrecurrent parent, while retaining essentially all of the rest ofthe desired genetic, and therefore the desired physiological andmorphological, constitution of the original inbred. The choice of theparticular nonrecurrent parent will depend on the purpose of thebackcross; one of the major purposes is to add some commerciallydesirable, agronomically important trait(s) to the plant. The exactbackcrossing protocol will depend on the characteristic(s) or trait(s)being altered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic beingtransferred is a single gene and dominant allele, multiple genes andrecessive allele(s) may also be transferred and therefore, backcrossbreeding is by no means restricted to character(s) governed by one or afew genes. In fact the number of genes might be less important that theidentification of the character(s) in the segregating population. Inthis instance it may then be necessary to introduce a test of theprogeny to determine if the desired characteristic(s) has beensuccessfully transferred. Such tests encompass visual inspection, simplecrossing, but also follow up of the characteristic(s) throughgenetically associated markers and molecular assisted breeding tools.For example, selection of progeny containing the transferred trait isdone by direct selection, visual inspection for a trait associated witha dominant allele, while the selection of progeny for a trait that istransferred via a recessive allele, such as the waxy starchcharacteristic in corn, require selfing the progeny to determine whichplant carry the recessive allele(s).

Many single gene traits have been identified that are not regularlyselected for in the development of a new parental inbred of a hybridBrassica rapa var. nipposinica plant according to the invention but thatcan be improved by backcrossing techniques. These genes are generallyinherited through the nucleus.

In 1981, the backcross method of breeding counted for 17% of the totalbreeding effort for inbred line development in the United States,accordingly to, Hallauer, A. R. et al. (1988) “Corn Breeding” Corn andCorn Improvement, No. 18, pp. 463-481.

The backcross breeding method provides a precise way of improvingvarieties that excel in a large number of attributes but are deficientin a few characteristics. (Page 150 of the Pr. R. W. Allard's 1960 book,published by John Wiley & Sons, Inc, Principles of Plant Breeding). Themethod makes use of a series of backcrosses to the variety to beimproved during which the character or the characters in whichimprovement is sought is maintained by selection. At the end of thebackcrossing the gene or genes being transferred unlike all other genes,will be heterozygous. Selfing after the last backcross produceshomozygosity for this gene pair(s) and, coupled with selection, willresult in a parental line of a hybrid variety with exactly theadaptation, yielding ability and quality characteristics of therecurrent parent but superior to that parent in the particularcharacteristic(s) for which the improvement program was undertaken.Therefore, this method provides the plant breeder with a high degree ofgenetic control of his work.

The method is scientifically exact because the morphological andagricultural features of the improved variety could be described inadvance and because the same variety could, if it were desired, be breda second time by retracing the same steps (Briggs, “Breeding wheatsresistant to bunt by the backcross method”, 1930 Jour. Amer. Soc.Agron., 22: 289-244).

Backcrossing is a powerful mechanism for achieving homozygosity and anypopulation obtained by backcrossing must rapidly converge on thegenotype of the recurrent parent. When backcrossing is made the basis ofa plant breeding program, the genotype of the recurrent parent will bemodified only with regards to genes being transferred, which aremaintained in the population by selection.

Successful backcrosses are, for example, the transfer of stem rustresistance from ‘Hope’ wheat to ‘Bart wheat’ and even pursuing thebackcrosses with the transfer of bunt resistance to create ‘Bart 38’,having both resistances. Also highlighted by Allard is the successfultransfer of mildew, leaf spot and wilt resistances in California Commonalfalfa to create ‘Caliverde’. This new ‘Caliverde’ variety producedthrough the backcross process is indistinguishable from CaliforniaCommon cxccpt for its resistance to the three named diseases.

One of the advantages of the backcross method is that the breedingprogram can be carried out in almost every environment that will allowthe development of the character being transferred.

The backcross technique is not only desirable when breeding for diseaseresistance but also for the adjustment of morphological characters,colour characteristics and simply inherited quantitative characters suchas earliness, plant height and seed size and shape. In this regard, amedium grain type variety, ‘Calady’, has been produced by Jones andDavis. As dealing with quantitative characteristics, they selected thedonor parent with the view of sacrificing some of the intensity of thecharacter for which it was chosen, i.e. grain size. ‘Lady Wright’, along grain variety was used as the donor parent and ‘Coloro’, a shortgrain one as the recurrent parent. After four backcrosses, the mediumgrain type variety ‘Calady’ was produced.

iii Single-Seed Descent and Multiple Seed Procedures

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F2 to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F2 individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F2 plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

In a multiple-seed procedure, breeders commonly harvest one or moreflower containing seed from each plant in a population and blend themtogether to form a bulk seed lot. Part of the bulked seed is used toplant the next generation and part is put in reserve. The procedure hasbeen referred to as modified single-seed descent or the bulk technique.

The multiple-seed procedure has been used to save labor at harvest. Itis considerably faster than removing one seed from each flower by handfor the single seed procedure. The multiple-seed procedure also makes itpossible to plant the same number of seeds of a population eachgeneration of inbreeding. Enough seeds are harvested to make up forthose plants that did not germinate or produce seed.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., R. W. Allard, 1960, Principles of Plant Breeding, JohnWiley and Son, pp. 115-161; N. W. Simmonds, 1979, Principles of CropImprovement, Longman Group Limited; W. R. Fehr, 1987, Principles of CropDevelopment, Macmillan Publishing Co.; N. F. Jensen, 1988, PlantBreeding Methodology, John Wiley & Sons).

iv Open-Pollinated Populations

The improvement of open-pollinated populations of such crops as rye,maize and sugar beets, herbage grasses, legumes such as alfalfa andclover, and tropical tree crops such as cacao, coconuts, oil palm andsome rubber, depends essentially upon changing gene-frequencies towardsfixation of favorable alleles while maintaining a high (but far frommaximal) degree of heterozygosity.

Uniformity in such populations is impossible and trueness-to-type in anopen-pollinated variety is a statistical feature of the population as awhole, not a characteristic of individual plants. Thus, theheterogeneity of open-pollinated populations contrasts with thehomogeneity (or virtually so) of inbred lines, clones and hybrids.

Population improvement methods fall naturally into two groups, thosebased on purely phenotypic selection, normally called mass selection,and those based on selection with progeny testing. Interpopulationimprovement utilizes the concept of open breeding populations; allowinggenes to flow from one population to another. Plants in one population(cultivar, strain, ecotype, or any germplasm source) are crossed eithernaturally (e.g., by wind) or by hand or by bees (commonly Apis melliferaL. or Megachile rotundata F.) with plants from other populations.Selection is applied to improve one (or sometimes both) population(s) byisolating plants with desirable traits from both sources.

There are basically two primary methods of open-pollinated populationimprovement.

First, there is the situation in which a population is changed en masseby a chosen selection procedure. The outcome is an improved populationthat is indefinitely propagable by random-mating within itself inisolation.

Second, the synthetic variety attains the same end result as populationimprovement, but is not itself propagable as such; it has to bereconstructed from parental lines or clones. These plant breedingprocedures for improving open-pollinated populations are well known tothose skilled in the art and comprehensive reviews of breedingprocedures routinely used for improving cross-pollinated plants areprovided in numerous texts and articles, including: Allard, Principlesof Plant Breeding, John Wiley & Sons, Inc. (1960); Simmonds, Principlesof Crop Improvement, Longman Group Limited (1979); Hallauer and Miranda,Quantitative Genetics in Maize Breeding, Iowa State University Press(1981); and, Jensen, Plant Breeding Methodology, John Wiley & Sons, Inc.(1988).

A) Mass Selection

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued. In massselection, desirable individual plants are chosen, harvested, and theseed composited without progeny testing to produce the followinggeneration. Since selection is based on the maternal parent only, andthere is no control over pollination, mass selection amounts to a formof random mating with selection. As stated above, the purpose of massselection is to increase the proportion of superior genotypes in thepopulation.

B) Synthetics

A synthetic variety is produced by crossing inter se a number ofgenotypes selected for good combining ability in all possible hybridcombinations, with subsequent maintenance of the variety by openpollination. Whether parents are (more or less inbred) seed-propagatedlines, as in some sugar beet and beans (Vicia) or clones, as in herbagegrasses, clovers and alfalfa, makes no difference in principle. Parentsare selected on general combining ability, sometimes by test crosses ortoperosses, more generally by polycrosses. Parental seed lines may bedeliberately inbred (e.g. by selling or sib crossing). However, even ifthe parents are not deliberately inbred, selection within lines duringline maintenance will ensure that some inbreeding occurs. Clonal parentswill, of course, remain unchanged and highly heterozygous.

Whether a synthetic can go straight from the parental seed productionplot to the farmer or must first undergo one or more cycles ofmultiplication depends on seed production and the scale of demand forseed. In practice, grasses and clovers are generally multiplied once ortwice and are thus considerably removed from the original synthetic.

While mass selection is sometimes used, progeny testing is generallypreferred for polycrosses, because of their operational simplicity andobvious relevance to the objective, namely exploitation of generalcombining ability in a synthetic.

The number of parental lines or clones that enters a synthetic varieswidely. In practice, numbers of parental lines range from 10 to severalhundred, with 100-200 being the average. Broad based synthetics formedfrom 100 or more clones would be expected to be more stable during seedmultiplication than narrow based synthetics.

iv. Hybrids

A hybrid is an individual plant resulting from a cross between parentsof differing genotypes. Commercial hybrids are now used extensively inmany crops, including corn (maize), sorghum, sugarbeet, sunflower andbroccoli. Hybrids can be formed in a number of different ways, includingby crossing two parents directly (single cross hybrids), by crossing asingle cross hybrid with another parent (three-way or triple crosshybrids), or by crossing two different hybrids (four-way or double crosshybrids).

Strictly speaking, most individuals in an out breeding (i.e.,open-pollinated) population are hybrids, but the term is usuallyreserved for cases in which the parents are individuals whose genomesare sufficiently distinct for them to be recognized as different speciesor subspecies. Hybrids may be fertile or sterile depending onqualitative and/or quantitative differences in the genomes of the twoparents. Heterosis, or hybrid vigor, is usually associated withincreased heterozygosity that results in increased vigor of growth,survival, and fertility of hybrids as compared with the parental linesthat were used to form the hybrid. Maximum heterosis is usually achievedby crossing two genetically different, highly inbred lines.

Once the inbreds that give the best hybrid performance have beenidentified, the hybrid seed can be reproduced indefinitely as long asthe homogeneity of the inbred parent is maintained. A single-crosshybrid is produced when two inbred lines are crossed to produce the F1progeny. A double-cross hybrid is produced from four inbred linescrossed in pairs (A×B and C×D) and then the two F1 hybrids are crossedagain (A×B)×(C×D). Much of the hybrid vigor and uniformity exhibited byF1 hybrids is lost in the next generation (F2). Consequently, seed fromF2 hybrid varieties is not used for planting stock.

The production of hybrids is a well-developed industry, involving theisolated production of both the parental lines and the hybrids whichresult from crossing those lines. For a detailed discussion of thehybrid production process, see, e.g., Wright, Commercial Hybrid SeedProduction 8:161-176, In Hybridization of Crop Plants.

v. Bulk Segregation Analysis (BSA)

BSA, a.k.a. bulked segregation analysis, or bulk segregant analysis, isa method described by Michelmore et al. (Michelmore et al., 1991,Identification of markers linked to disease-resistance genes by bulkedsegregant analysis: a rapid method to detect markers in specific genomicregions by using segregating populations. Proceedings of the NationalAcademy of Sciences, USA, 99:9828-9832) and Quarrie et al. (Quarrie etal., 1999, Journal of Experimental Botany, 50(337):1299-1306).

For BSA of a trait of interest, parental lines with certain differentphenotypes are chosen and crossed to generate F2, doubled haploid orrecombinant inbred populations with QTL analysis. The population is thenphenotyped to identify individual plants or lines having high or lowexpression of the trait. Two DNA bulks are prepared, one from theindividuals having one phenotype (e.g., resistant to virus), and theother from the individuals having reversed phenotype (e.g., susceptibleto virus), and analyzed for allele frequency with molecular markers.Only a few individuals are required in each bulk (e.g., 10 plants each)if the markers are dominant (e.g., RAPDs). More individuals are neededwhen markers are co-dominant (e.g., RFLPs). Markers linked to thephenotype can be identified and used for breeding or QTL mapping.

vi. Hand-Pollination Method

Hand pollination describes the crossing of plants via the deliberatefertilization of female ovules with pollen from a desired male parentplant. In some embodiments the donor or recipient female parent and thedonor or recipient male parent line are planted in the same field. Insome embodiments the donor or recipient female parent and the donor orrecipient male parent line are planted in the same greenhouse. Theinbred male parent can be planted earlier than the female parent toensure adequate pollen supply at the pollination time. In someembodiments, the male parent and female parent can be planted at a ratioof 1 male parent to 4-10 female parents. Pollination is started when thefemale parent flower is ready to be fertilized. Female flower buds thatare ready to open in the following days are identified, covered withpaper cups or small paper bags that prevent bee or any other insect fromvisiting the female flowers, and marked with any kind of material thatcan be easily seen the next morning. The male flowers of the male parentare collected in the early morning before they are open and visited bypollinating insects. The covered female flowers of the female parent,which have opened, are un-covered and pollinated with the collectedfresh male flowers of the male parent, starting as soon as the maleflower sheds pollen. The pollinated female flowers are again coveredafter pollination to prevent bees and any other insects visit. Thepollinated female flowers are also marked. The marked flowers areharvested. In some embodiments, the male pollen used for fertilizationhas been previously collected and stored.

vii. Bee-Pollination Method

Using the bee-pollination method, the parent plants are usually plantedwithin close proximity. In some embodiments more female plants areplanted to allow for a greater production of seed. Insects are placed inthe field or greenhouses for transfer of pollen from the male parent tothe female flowers of the female parent.

viii. Targeting Induced Local Lesions in Genomes (TILLING)

Breeding schemes of the present application can include crosses withTILLING® plant cultivars. TILLING® is a method in molecular biology thatallows directed identification of mutations in a specific gene. TILLING®was introduced in 2000, using the model plant Arabidopsis thaliana.TILLING® has since been used as a reverse genetics method in otherorganisms such as zebrafish, corn, wheat, rice, soybean, tomato andBrassica rapa var. nipposinica.

The method combines a standard and efficient technique of mutagenesiswith a chemical mutagen (e.g., Ethyl methanesulfonate (EMS)) with asensitive DNA screening-technique that identifies single base mutations(also called point mutations) in a target gene. EcoTILLING is a methodthat uses TILLING® techniques to look for natural mutations inindividuals, usually for population genetics analysis (see Comai, etal., 2003 The Plant Journal 37, 778-786; Gilchrist et al. 2006 Mol.Ecol. 15, 1367-1378; Mejlhede et al. 2006 Plant Breeding 125, 461-467;Nieto et al. 2007 BMC Plant Biology 7, 34-42, each of which isincorporated by reference hereby for all purposes). DEcoTILLING is amodification of TILLING® and EcoTILLING which uses an inexpensive methodto identify fragments (Garvin et al., 2007, DEco-TILLING: An inexpensivemethod for SNP discovery that reduces ascertainment bias. MolecularEcology Notes 7, 735-746).

The TILLING® method relies on the formation of heteroduplexes that areformed when multiple alleles (which could be from a heterozygote or apool of multiple homozygotes and heterozygotes) are amplified in a PCR,heated, and then slowly cooled. A “bubble” forms at the mismatch of thetwo DNA strands (the induced mutation in TILLING® or the naturalmutation or SNP in EcoTILLING), which is then cleaved by single strandednucleases. The products are then separated by size on several differentplatforms.

Several TILLING® centers exists over the world that focus onagriculturally important species: UC Davis (USA), focusing on Rice;Purdue University (USA), focusing on Maize; University of BritishColumbia (CA), focusing on Brassica napus; John Innes Centre (UK),focusing on Brassica rapa; Fred Hutchinson Cancer Research, focusing onArabidopsis; Southern Illinois University (USA), focusing on Soybean;John Innes Centre (UK), focusing on Lotus and Medicago; and INRA(France), focusing on Pea and Tomato.

More detailed description on methods and compositions on TILLING® can befound in U.S. Pat. No. 5,994,075, US 2004/0053236 A1, WO 2005/055704,and WO 2005/048692, each of which is hereby incorporated by referencefor all purposes.

Thus in some embodiments, the breeding methods of the present disclosureinclude breeding with one or more TILLING plant lines with one or moreidentified mutations.

viii Mutation Breeding

Mutation breeding is another method of introducing new traits intoplants. Mutations that occur spontaneously or are artificially inducedcan be useful sources of variability for a plant breeder. The goal ofartificial mutagenesis is to increase the rate of mutation for a desiredcharacteristic. Mutation rates can be increased by many different meansor mutating agents including temperature, long-term seed storage, tissueculture conditions, radiation (such as X-rays, Gamma rays, neutrons,Beta radiation, or ultraviolet radiation), chemical mutagens (such asbase analogs like 5-bromo-uracil), antibiotics, alkylating agents (suchas sulfur mustards, nitrogen mustards, epoxides, ethyleneamines,sulfates, sulfonates, sulfones, or lactones), azide, hydroxylamine,nitrous acid or acridines. Once a desired trait is observed throughmutagenesis the trait may then be incorporated into existing germplasmby traditional breeding techniques. Details of mutation breeding can befound in W. R. Fehr, 1993, Principles of Cultivar Development, MacmillanPublishing Co.

New breeding techniques such as the ones involving the uses of ZincFinger Nucleases or oligonucleotide directed mutagenesis shall also beused to generate genetic variability and introduce new traits intovarieties.

ix. Double Haploids and Chromosome Doubling

One way to obtain homozygous plants without the need to cross twoparental lines followed by a long selection of the segregating progeny,and/or multiple back-crossings is to produce haploids and then doublethe chromosomes to form doubled haploids. Haploid plants can occurspontaneously, or may be artificially induced via chemical treatments orby crossing plants with inducer lines (Seymour et al. 2012, PNAS vol109, pg 4227-4232; Zhang et al., 2008 Plant Cell Rep. December 27(12)1851-60). The production of haploid progeny can occur via a variety ofmechanisms which can affect the distribution of chromosomes duringgamete formation. The chromosome complements of haploids sometimesdouble spontaneously to produce homozygous doubled haploids (DHs).Mixoploids, which are plants which contain cells having differentploidies, can sometimes arise and may represent plants that areundergoing chromosome doubling so as to spontaneously produce doubledhaploid tissues, organs, shoots, floral parts or plants. Another commontechnique is to induce the formation of double haploid plants with achromosome doubling treatment such as colchicine (El-Hennawy et al.,2011 Vol 56, issue 2 pg 63-72; Doubled Haploid Production in Crop Plants2003 edited by Maluszynski ISBN 1-4020-1544-5). The production ofdoubled haploid plants yields highly uniform cultivars and is especiallydesirable as an alternative to sexual inbreeding of longer-generationcrops. By producing doubled haploid progeny, the number of possible genecombinations for inherited traits is more manageable. Thus, an efficientdoubled haploid technology can significantly reduce the time and thecost of inbred and cultivar development.

x. Protoplast Fusion

In another method for breeding plants, protoplast fusion can also beused for the transfer of trait-conferring genomic material from a donorplant to a recipient plant. Protoplast fusion is an induced orspontaneous union, such as a somatic hybridization, between two or moreprotoplasts (cells of which the cell walls are removed by enzymatictreatment) to produce a single bi- or multi-nucleate cell. The fusedcell that may even be obtained with plant species that cannot beinterbred in nature is tissue cultured into a hybrid plant exhibitingthe desirable combination of traits.

xi. Embryo Rescue

Alternatively, embryo rescue may be employed in the transfer ofresistance-conferring genomic material from a donor plant to a recipientplant. Embryo rescue can be used as a procedure to isolate embryo's fromcrosses wherein plants fail to produce viable seed. In this process, thefertilized ovary or immature seed of a plant is tissue cultured tocreate new plants (see Pierik, 1999, In vitro culture of higher plants,Springer, ISBN 079235267x, 9780792352679, which is incorporated hereinby reference in its entirety).

Breeding Evaluation

Each breeding program can include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested per se and inhybrid combination and compared to appropriate standards in environmentsrepresentative of the commercial target area(s). The best lines arecandidates for use as parents in new commercial cultivars; those stilldeficient in a few traits may be used as parents to produce newpopulations for further selection.

In one embodiment, the plants are selected on the basis of one or morephenotypic traits. Skilled persons will readily appreciate that suchtraits include any observable characteristic of the plant, including forexample growth rate, height, weight, color, taste, smell, changes in theproduction of one or more compounds by the plant (including for example,metabolites, proteins, drugs, carbohydrates, oils, and any othercompounds).

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor and consumer; for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

It should be appreciated that in certain embodiments, plants may beselected based on the absence, suppression or inhibition of a certainfeature or trait (such as an undesirable feature or trait) as opposed tothe presence of a. certain feature or trait (such as a desirable featureor trait).

Selecting plants based on genotypic information is also envisaged (forexample, including the pattern of plant gene expression, genotype, orpresence of genetic markers). Where the presence of one or more geneticmarker is assessed, the one or more marker may already be known and/orassociated with a particular characteristic of a plant; for example, amarker or markers may be associated with an increased growth rate ormetabolite profile. This information could be used in combination withassessment based on other characteristics in a method of the disclosureto select for a combination of different plant characteristics that maybe desirable. Such techniques may be used to identify novel quantitativetrait loci (QTLs). By way of example, plants may be selected based ongrowth rate, size (including but not limited to weight, height, leafsize, stem size, branching pattern, or the size of any part of theplant), general health, survival, tolerance to adverse physicalenvironments and/or any other characteristic, as described hereinbefore.

Further non-limiting examples include selecting plants based on: speedof seed germination; quantity of biomass produced; increased root,and/or leaf/shoot growth that leads to an increased yield (herbage orgrain or fiber or oil) or biomass production; effects on plant growththat results in an increased seed yield for a crop; effects on plantgrowth which result in an increased yield; effects on plant growth thatlead to an increased resistance or tolerance disease including fungal,viral or bacterial diseases or to pests such as insects, mites ornematodes in which damage is measured by decreased foliar symptoms suchas the incidence of bacterial or fungal lesions, or area of damagedfoliage or reduction in the numbers of nematode cysts or galls on plantroots, or improvements in plant yield in the presence of such plantpests and diseases; effects on plant growth that lead to increasedmetabolite yields; effects on plant growth that lead to improvedaesthetic appeal which may be particularly important in plants grown fortheir form, color or taste, for example the color intensity of Brassicarapa var. nipposinica leaves, or the taste of said leaves.

Molecular Breeding Evaluation Techniques

Selection of plants based on phenotypic or genotypic information may beperformed using techniques such as, but not limited to: high through-putscreening of chemical components of plant origin, sequencing techniquesincluding high through-put sequencing of genetic material, differentialdisplay techniques (including DDRT-PCR, and DD-PCR), nucleic acidmicroarray techniques, RNA-seq (Whole Transcriptome Shotgun Sequencing),qRTPCR (quantitative real time PCR).

In one embodiment, the evaluating step of a plant breeding programinvolves the identification of desirable traits in progeny plants.Progeny plants can be grown in, or exposed to conditions designed toemphasize a particular trait (e.g. drought conditions for droughttolerance, lower temperatures for freezing tolerant traits). Progenyplants with the highest scores for a particular trait may be used forsubsequent breeding steps.

In some embodiments, plants selected from the evaluation step canexhibit a 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 120% or more improvement in aparticular plant trait compared to a control plant.

In other embodiments, the evaluating step of plant breeding comprisesone or more molecular biological tests for genes or other markers. Forexample, the molecular biological test can involve probe hybridizationand/or amplification of nucleic acid (e.g., measuring nucleic aciddensity by Northern or Southern hybridization, PCR) and/or immunologicaldetection (e.g., measuring protein density, such as precipitation andagglutination tests, ELISA (e.g., Lateral Flow test or DAS-ELISA),Western blot, RIA, immune labeling, immunosorbent electron microscopy(ISEM), and/or dot blot).

The procedure to perform a nucleic acid hybridization, an amplificationof nucleic acid (e.g., RT-PCR) or an immunological detection (e.g.,precipitation and agglutination tests, ELISA (e.g., Lateral Flow test orDAS-ELISA), Western blot, RIA, immunogold or immunofluorescent labeling,immunosorbent electron microscopy (ISEM), and/or dot blot tests) areperformed as described elsewhere herein and well-known by one skilled inthe art.

In one embodiment, the evaluating step comprises PCR (semi-quantitativeor quantitative), wherein primers are used to amplify one or morenucleic acid sequences of a desirable gene, or a nucleic acid associatedwith said gene or a desirable trait (e.g., a co-segregating nucleicacid, or other marker).

In another embodiment, the evaluating step comprises immunologicaldetection (e.g., precipitation and agglutination tests, ELISA (e.g.,Lateral Flow test or DAS-ELISA), Western blot, RIA, immuno labeling(gold, fluorescent, or other detectable marker), immunosorbent electronmicroscopy (ISEM), and/or dot blot), wherein one or more gene ormarker-specific antibodies are used to detect one or more desirableproteins. In one embodiment, said specific antibody is selected from thegroup consisting of polyclonal antibodies, monoclonal antibodies,antibody fragments, and combination thereof.

Reverse Transcription Polymerase Chain Reaction (RT-PCR) can be utilizedin the present disclosure to determine expression of a gene to assistduring the selection step of a breeding scheme. It is a variant ofpolymerase chain reaction (PCR), a laboratory technique commonly used inmolecular biology to generate many copies of a DNA sequence, a processtermed “amplification”. In RT-PCR, however, RNA strand is first reversetranscribed into its DNA complement (complementary DNA, or cDNA) usingthe enzyme reverse transcriptase, and the resulting cDNA is amplifiedusing traditional or real-time PCR.

RT-PCR utilizes a pair of primers, which are complementary to a definedsequence on each of the two strands of the cDNA. These primers are thenextended by a DNA polymerase and a copy of the strand is made after eachcycle, leading to logarithmic amplification.

RT-PCR includes three major steps. The first step is the reversetranscription (RT) where RNA is reverse transcribed to cDNA using areverse transcriptase and primers. This step is very important in orderto allow the performance of PCR since DNA polymerase can act only on DNAtemplates. The RT step can be performed either in the same tube with PCR(one-step PCR) or in a separate one (two-step PCR) using a temperaturebetween 40° C. and 50° C., depending on the properties of the reversetranscriptase used.

The next step involves the denaturation of the dsDNA at 95° C., so thatthe two strands separate and the primers can bind again at lowertemperatures and begin a new chain reaction. Then, the temperature isdecreased until it reaches the annealing temperature which can varydepending on the set of primers used, their concentration, the probe andits concentration (if used), and the cations concentration. The mainconsideration, of course, when choosing the optimal annealingtemperature is the melting temperature (Tm) of the primers and probes(if used). The annealing temperature chosen for a PCR depends directlyon length and composition of the primers. This is the result of thedifference of hydrogen bonds between A-T (2 bonds) and G-C (3 bonds). Anannealing temperature about 5 degrees below the lowest Tm of the pair ofprimers is usually used.

The final step of PCR amplification is the DNA extension from theprimers which is done by the thermostable Taq DNA polymerase usually at72° C., which is the optimal temperature for the polymerase to work. Thelength of the incubation at each temperature, the temperaturealterations and the number of cycles are controlled by a programmablethermal cycler. The analysis of the PCR products depends on the type ofPCR applied. If a conventional PCR is used, the PCR product is detectedusing agarose gel electrophoresis and ethidium bromide (or other nucleicacid staining).

Conventional RT-PCR is a time-consuming technique with importantlimitations when compared to real time PCR techniques. This, combinedwith the fact that ethidium bromide has low sensitivity, yields resultsthat are not always reliable. Moreover, there is an increasedcross-contamination risk of the samples since detection of the PCRproduct requires the post-amplification processing of the samples.Furthermore, the specificity of the assay is mainly determined by theprimers, which can give false-positive results. However, the mostimportant issue concerning conventional RT-PCR is the fact that it is asemi or even a low quantitative technique, where the amplicon can bevisualized only after the amplification ends.

Real time RT-PCR provides a method where the amplicons can be visualizedas the amplification progresses using a fluorescent reporter molecule.There are three major kinds of fluorescent reporters used in real timeRT-PCR, general non specific DNA Binding Dyes such as SYBR Green I,TaqMan Probes and Molecular Beacons (including Scorpions).

The real time PCR thermal cycler has a fluorescence detection threshold,below which it cannot discriminate the difference between amplificationgenerated signal and background noise. On the other hand, thefluorescence increases as the amplification progresses and theinstrument performs data acquisition during the annealing step of eachcycle. The number of amplicons will reach the detection baseline after aspecific cycle, which depends on the initial concentration of the targetDNA sequence. The cycle at which the instrument can discriminate theamplification generated fluorescence from the background noise is calledthe threshold cycle (Ct). The higher is the initial DNA concentration,the lower its Ct will be.

Other forms of nucleic acid detection can include next generationsequencing methods such as DNA SEQ or RNA SEQ using any known sequencingplatform including, but not limited to: Roche 454, Solexa GenomeAnalyzer, AB SOLiD, Illumina GA/HiSeq, Ion PGM, Mi Seq, among others(Liu et al., 2012 Journal of Biomedicine and Biotechnology Volume 2012ID 251364; Franca et al., 2002 Quarterly Reviews of Biophysics 35 pg169-200; Mardis 2008 Genomics and Human Genetics vol 9 pg 387-402).

In other embodiments, nucleic acids may be detected with other highthroughput hybridization technologies including microarrays, gene chips,LNA probes, nanoStrings, and fluorescence polarization detection amongothers.

In some embodiments, detection of markers can be achieved at an earlystage of plant growth by harvesting a small tissue sample (e.g., branch,or leaf disk). This approach is preferable when working with largepopulations as it allows breeders to weed out undesirable progeny at anearly stage and conserve growth space and resources for progeny whichshow more promise. In some embodiments the detection of markers isautomated, such that the detection and storage of marker data is handledby a machine. Recent advances in robotics have also led to full serviceanalysis tools capable of handling nucleic acid/protein markerextractions, detection, storage and analysis.

Quantitative Trait Loci

Breeding schemes of the present application can include crosses betweendonor and recipient plants. In some embodiments said donor plantscontain a gene or genes of interest which may confer the plant with adesirable phenotype. The recipient line can be an elite line or cultivarhaving certain favorite traits such for commercial production. In oneembodiment, the elite line may contain other genes that also impart saidline with the desired phenotype. When crossed together, the donor andrecipient plant may create a progeny plant with combined desirable lociwhich may provide quantitatively additive effect of a particularcharacteristic. In that case, QTL mapping can be involved to facilitatethe breeding process.

A QTL (quantitative trait locus) mapping can be applied to determine theparts of the donor plant's genome conferring the desirable phenotype,and facilitate the breeding methods. Inheritance of quantitative traitsor polygenic inheritance refers to the inheritance of a phenotypiccharacteristic that varies in degree and can be attributed to theinteractions between two or more genes and their environment. Though notnecessarily genes themselves, quantitative trait loci (QTLs) arestretches of DNA that are closely linked to the genes that underlie thetrait in question. QTLs can be molecularly identified to help mapregions of the genome that contain genes involved in specifying aquantitative trait. This can be an early step in identifying andsequencing these genes.

Typically, QTLs underlie continuous traits (those traits that varycontinuously, e.g. yield, height, level of resistance to virus, etc.) asopposed to discrete traits (traits that have two or several charactervalues, e.g. smooth vs. wrinkled peas used by Mendel in hisexperiments). Moreover, a single phenotypic trait is usually determinedby many genes. Consequently, many QTLs are associated with a singletrait.

A quantitative trait locus (QTL) is a region of DNA that is associatedwith a particular phenotypic trait—these QTLs are often found ondifferent chromosomes. Knowing the number of QTLs that explainsvariation in the phenotypic trait tells about the genetic architectureof a trait. It may tell that a trait is controlled by many genes ofsmall effect, or by a few genes of large effect.

Another use of QTLs is to identify candidate genes underlying a trait.Once a region of DNA is identified as contributing to a phenotype, itcan be sequenced. The DNA sequence of any genes in this region can thenbe compared to a database of DNA for genes whose function is alreadyknown.

In a recent development, classical QTL analyses are combined with geneexpression profiling i.e. by DNA microarrays. Such expression QTLs(e-QTLs) describes cis- and trans-controlling elements for theexpression of often disease-associated genes. Observed epistatic effectshave been found beneficial to identify the gene responsible by across-validation of genes within the interacting loci with metabolicpathway- and scientific literature databases.

QTL mapping is the statistical study of the alleles that occur in alocus and the phenotypes (physical forms or traits) that they produce(see, Meksem and Kahl, The handbook of plant genome mapping: genetic andphysical mapping, 2005, Wiley-VCH, ISBN 3527311165, 9783527311163).Because most traits of interest are governed by more than one gene,defining and studying the entire locus of genes related to a trait giveshope of understanding what effect the genotype of an individual mighthave in the real world.

Statistical analysis is required to demonstrate that different genesinteract with one another and to determine whether they produce asignificant effect on the phenotype. QTLs identify a particular regionof the genome as containing a gene that is associated with the traitbeing assayed or measured. They are shown as intervals across achromosome, where the probability of association is plotted for eachmarker used in the mapping experiment.

To begin, a set of genetic markers must be developed for the species inquestion. A marker is an identifiable region of variable DNA. Biologistsare interested in understanding the genetic basis of phenotypes(physical traits). The aim is to find a marker that is significantlymore likely to co-occur with the trait than expected by chance, that is,a marker that has a statistical association with the trait. Ideally,they would be able to find the specific gene or genes in question, butthis is a long and difficult undertaking. Instead, they can more readilyfind regions of DNA that are very close to the genes in question. When aQTL is found, it is often not the actual gene underlying the phenotypictrait, but rather a region of DNA that is closely linked with the gene.

For organisms whose genomes are known, one might now try to excludegenes in the identified region whose function is known with somecertainty not to be connected with the trait in question. If the genomeis not available, it may be an option to sequence the identified regionand determine the putative functions of genes by their similarity togenes with known function, usually in other genomes. This can be doneusing BLAST, an online tool that allows users to enter a primarysequence and search for similar sequences within the BLAST database ofgenes from various organisms.

Another interest of statistical geneticists using QTL mapping is todetermine the complexity of the genetic architecture underlying aphenotypic trait. For example, they may be interested in knowing whethera phenotype is shaped by many independent loci, or by a few loci, and dothose loci interact. This can provide information on how the phenotypemay be evolving.

Molecular markers are used for the visualization of differences innucleic acid sequences. This visualization is possible due to DNA-DNAhybridization techniques (RFLP) and/or due to techniques using thepolymerase chain reaction (e.g. STS, microsatellites, AFLP). Alldifferences between two parental genotypes will segregate in a mappingpopulation based on the cross of these parental genotypes. Thesegregation of the different markers may be compared and recombinationfrequencies can be calculated. The recombination frequencies ofmolecular markers on different chromosomes are generally 50%. Betweenmolecular markers located on the same chromosome the recombinationfrequency depends on the distance between the markers. A lowrecombination frequency corresponds to a low distance between markers ona chromosome. Comparing all recombination frequencies will result in themost logical order of the molecular markers on the chromosomes. Thismost logical order can be depicted in a linkage map (Paterson, 1996,Genome Mapping in Plants. R. G. Landes, Austin.). A group of adjacent orcontiguous markers on the linkage map that is associated to a reduceddisease incidence and/or a reduced lesion growth rate pinpoints theposition of a QTL.

The nucleic acid sequence of a QTL may be determined by methods known tothe skilled person. For instance, a nucleic acid sequence comprisingsaid QTL or a resistance-conferring part thereof may be isolated from adonor plant by fragmenting the genome of said plant and selecting thosefragments harboring one or more markers indicative of said QTL.Subsequently, or alternatively, the marker sequences (or parts thereof)indicative of said QTL may be used as (PCR) amplification primers, inorder to amplify a nucleic acid sequence comprising said QTL from agenomic nucleic acid sample or a genome fragment obtained from saidplant. The amplified sequence may then be purified in order to obtainthe isolated QTL. The nucleotide sequence of the QTL, and/or of anyadditional markers comprised therein, may then be obtained by standardsequencing methods.

One or more such QTLs associated with a desirable trait in a donor plantcan be transferred to a recipient plant to make incorporate thedesirable train into progeny plants by transferring and/or breedingmethods.

In one embodiment, an advanced backcross QTL analysis (AB-QTL) is usedto discover the nucleotide sequence or the QTLs responsible for theresistance of a plant. Such method was proposed by Tanksley and Nelsonin 1996 (Tanksley and Nelson, 1996, Advanced backcross QTL analysis: amethod for simultaneous discovery and transfer of valuable QTL fromun-adapted germplasm into elite breeding lines. Theor Appl Genet92:191-203) as a new breeding method that integrates the process of QTLdiscovery with variety development, by simultaneously identifying andtransferring useful QTL alleles from un-adapted (e.g., land races, wildspecies) to elite germplasm, thus broadening the genetic diversityavailable for breeding. AB-QTL strategy was initially developed andtested in tomato, and has been adapted for use in other crops includingrice, maize, wheat, pepper, barley, and bean. Once favorable QTL allelesare detected, only a few additional marker-assisted generations arerequired to generate near isogenic lines (NILs) or introgression lines(ILs) that can be field tested in order to confirm the QTL effect andsubsequently used for variety development.

Isogenic lines in which favorable QTL alleles have been fixed can begenerated by systematic backcrossing and introgressing of marker-defineddonor segments in the recurrent parent background. These isogenic linesare referred as near isogenic lines (NILs), introgression lines (ILs),backcross inbred lines (BILs), backcross recombinant inbred lines(BCRIL), recombinant chromosome substitution lines (RCSLs), chromosomesegment substitution lines (CSSLs), and stepped aligned inbredrecombinant strains (STAIRSs). An introgression line in plant molecularbiology is a line of a crop species that contains genetic materialderived from a similar species. ILs represent NILs with relatively largeaverage introgression length, while BILs and BCRILs are backcrosspopulations generally containing multiple donor introgressions per line.As used herein, the term “introgression lines or ILs” refers to plantlines containing a single marker defined homozygous donor segment, andthe term “pre-ILs” refers to lines which still contain multiplehomozygous and/or heterozygous donor segments.

To enhance the rate of progress of introgression breeding, a geneticinfrastructure of exotic libraries can be developed. Such an exoticlibrary comprises of a set of introgression lines, each of which has asingle, possibly homozygous, marker-defined chromosomal segment thatoriginates from a donor exotic parent, in an otherwise homogenous elitegenetic background, so that the entire donor genome would be representedin a set of introgression lines. A collection of such introgressionlines is referred as libraries of introgression lines or IL libraries(ILLs). The lines of an ILL cover usually the complete genome of thedonor, or the part of interest. Introgression lines allow the study ofquantitative trait loci, but also the creation of new varieties byintroducing exotic traits. High resolution mapping of QTL using ILLsenable breeders to assess whether the effect on the phenotype is due toa single QTL or to several tightly linked QTL affecting the same trait.In addition, sub-ILs can be developed to discover molecular markerswhich are more tightly linked to the QTL of interest, which can be usedfor marker-assisted breeding (MAB). Multiple introgression lines can bedeveloped when the introgression of a single QTL is not sufficient toresult in a substantial improvement in agriculturally important traits(Gur and Zamir, Unused natural variation can lift yield barriers inplant breeding, 2004, PLoS Biol.; 2(10):e245).

Tissue Culture

As it is well known in the art, tissue culture of Brassica rapa var.nipposinica can be used for the in vitro regeneration of Brassica rapavar. nipposinica plants. Tissues cultures of various tissues of Brassicarapa var. nipposinica and regeneration of plants therefrom are wellknown. Thus, another aspect of this invention is to provide cells whichupon growth and differentiation produce Brassica rapa var. nipposinicaplants having the physiological and morphological characteristics ofBrassica rapa var. nipposinica cultivar ORIGAMI.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, plant clumps, and plantcells that can generate tissue culture that are intact in plants orparts of plants, such as embryos, pollen, flowers, seeds, leaves, stems,roots, root tips, anthers, pistils, meristematic cells, axillary buds,ovaries, seed coat, endosperm, hypocotyls, cotyledons and the like.Means for preparing and maintaining plant tissue culture are well knownin the art. By way of example, a tissue culture comprising organs hasbeen used to produce regenerated plants. U.S. Pat. Nos. 5,959,185,5,973,234, and 5,977,445 describe certain techniques, the disclosures ofwhich are incorporated herein by reference.

EXAMPLES

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

Example 1—Development of Brassica rapa var. nipposinica cultivar ORIGAMI

The Brassica rapa var. nipposinica cultivar ORIGAMI is a dark greenBrassica rapa var. nipposinica variety obtained after several rounds ofself-pollination and selection.

Breeding History:

ORIGAMI was developed from a single unusual plant discovered growing inan experimental field trial in Salinas on May of the first year. Theplant was added to a trial of assorted baby leaf material, and the seedproduced was designated source code 40-0701-231-1.

The resulting seed was grown in spring of the second year at Gilroy.Three selections were made and allowed to self, and the seed of eachplant was collected individually. The three batches of seed wereassigned the source codes B-811-92-1, -2, and -3. The resulting seed wasgrown in autumn of the second at Gilroy. One selection was made fromB-811-92-3 and allowed to self and the seeds collected. The seed wasassigned the source code B-829-16-1. The resulting seed was grown inautumn of year three at Gilroy. Three selections were made and allowedto self, and the seed of each plant was collected individually. Thethree batches of seed were assigned the source codes B-915-20-1, -2, and-3. The resulting seed was grown in autumn of year four at Gilroy. Eightselections were made from B-915-20-3 and allowed to self, and the seedof each plant was collected individually. The eight batches of seed wereassigned the source codes B-1034-34-1, -2, -3, -4, -5, -6, -7, and -8.The resulting seed was grown in autumn of year five at Gilroy. Theplants of B-1034-34-5 were planted in a seed increase cage. All of theplants were harvested in bulk and those seeds became ORIGAMI.

Some of the criteria used to select the Brassica rapa var. nipposinicacultivar ORIGAMI in various generations include: uniformity, color ofthe leaves, texture of the leaves, extension of leaf blade, length ofhypocotyl and frilliness, i.e. the ratio of length of leaf division tothe size of leaf and mount of subdivision.

ORIGAMI Brassica rapa var. nipposinica cultivar similar to the Mizunavariety sold by Shamrock Seed Company, but has numerous differences:ORIGAMI leaf color is RHS green 137C while Mizuna is RHS green 137B,with heavier texture and narrower leaves, and is more uniform. The leafblades extend less far from the petioles and secondary veins so that thedivisions are more incised, and it resists making fan-shaped leaves. Theleaves are less frilly and it is slightly less vigorous than Mizunavariety.

The Brassica rapa var. nipposinica cultivar ORIGAMI has shown uniformityand stability for the traits, within the limits of environmentalinfluence for the traits as described in the following VarietyDescriptive Information. No variant traits have been observed or areexpected for agronomical important traits in Brassica rapa var.nipposinica cultivar ORIGAMI.

Brassica rapa var. nipposinica cultivar ORIGAMI has the followingmorphologic and other characteristics, (based primarily on datacollected in California, all experiments done under the directsupervision of the applicant).

TABLE 1 Variety Description Information Plant: Brassica rapa var.nipposinica Seed: Color: Dark Brown Cotyledon to Fourth Leaf Stage:Shape of cotyledon: Reniform Shape of fourth leaf: Elongated ApicalMargin: Divided Basal Margin: Divided Undulation: None Green Color:Medium Green Anthocyanin Distribution: Absent Anthocyanin Concentration:N/A Rolling (curvature parallel to the spine of the Absent leaf):Cupping: Uncupped Reflexing (curvature perpendicular to the spine Slightof the leaf): Harvest-Mature Out Leaf, Head, Core: Margin IncisionDepth: Deep Margin Indentation: Deeply Dentate Undulation of the ApicalMargin: None Green Color: Medium green Anthocyanin Distribution: AbsentAnthocyanin Concentration: N/A Anthocyanin Size: N/A Glossiness:Moderate Blistering: None Leaf Thickness: Medium Thick Trichomes: Absent(Smooth) Maturity (No. of Days of First Water date to 24 Harvest): OuterLeaf Length (cm): full size harvest 20.4 Out Leaf Width (cm): full sizeharvest 6.5 Outer Leaf Length (cm): baby leaf size harvest 10.2 Out LeafWidth (cm): baby leaf size harvest 4.5 Adaptation: Primary Regions ofAdaptation (tested and proven adapted): Costal California and SouthwestDesert (California and Arizona) Season: Spring to autumn in CostalCalifornia and Autumn to Spring in the Desert Southwest. Warm to coldtransition for Yuma

DEPOSIT INFORMATION

A deposit of the Brassica rapa var. nipposinica seed of this inventionis maintained by Shamrock Seed Company Inc., 3 Harris Place, Salinas,Calif. 93901-4593, USA. In addition, a sample of the Brassica rapa var.nipposinica seed of this invention has been deposited with the NationalCollections of Industrial, Food and Marine Bacteria (NCIMB), 23 StMachar Drive, Aberdeen, Scotland, AB24 3RY, United Kingdom.

To satisfy the enablement requirements of 35 U.S.C. 112, and to certifythat the deposit of the isolated strain of the present invention meetsthe criteria set forth in 37 C.F.R. 1.801-1.809, Applicants hereby makethe following statements regarding the deposited Brassica rapa var.nipposinica cultivar ORIGAMI (deposited as NCIMB Accession No. ______):

1. During the pendency of this application, access to the invention willbe afforded to the Commissioner upon request;2. All restrictions on availability to the public will be irrevocablyremoved upon granting of the patent under conditions specified in 37 CFR1.808;3. The deposit will be maintained in a public repository for a period of30 years or 5 years after the last request or for the effective life ofthe patent, whichever is longer;4. A test of the viability of the biological material at the time ofdeposit will be conducted by the public depository under 37 C.F.R.1.807; and5. The deposit will be replaced if it should ever become unavailable.Access to this deposit will be available during the pendency of thisapplication to persons determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. Upon allowance of any claims in this application, all restrictionson the availability to the public of the variety will be irrevocablyremoved by affording access to a deposit of at least 2,500 seeds of thesame variety with the NCIMB.

INCORPORATION BY REFERENCE

. All references, articles, publications, patents, patent publications,and patent applications cited herein are incorporated by reference intheir entireties for all purposes.

However, mention of any reference, article, publication, patent, patentpublication, and patent application cited herein is not, and should notbe taken as an acknowledgment or any form of suggestion that theyconstitute valid prior art or form part of the common general knowledgein any country in the world.

What is claimed is:
 1. A seed of Brassica rapa var. nipposinicadesignated ORIGAMI, wherein a representative sample of seed of saidBrassica rapa var. nipposinica having been deposited under NCIMB No______.
 2. A Brassica rapa var. nipposinica plant, or a part or a plantcell thereof, produced by growing the seed of claim
 1. 3. The Brassicarapa var. nipposinica part of claim 2, wherein the Brassica rapa var.nipposinica part is selected from the group consisting of: a leaf, aflower, an ovule, a pollen and a cell.
 4. A Brassica rapa var.nipposinica plant, having all of the characteristics of Brassica rapavar. nipposinica ORIGAMI listed in Table 1 when grown in the sameenvironmental conditions, or a part or a plant cell thereof.
 5. ABrassica rapa var. nipposinica plant, or a part thereof, having all ofthe physiological and morphological characteristics of Brassica rapavar. nipposinica ORIGAMI, wherein a representative sample of seed ofsaid Brassica rapa var. nipposinica having been deposited under NCIMB No______.
 6. A tissue culture of regenerable cells produced from the plantor plant part of claim 2, wherein a plant regenerated from the tissueculture has all of the characteristics of Brassica rapa var. nipposinicaORIGAMI listed in Table 1 when grown in the same environmentalconditions.
 7. A Brassica rapa var. nipposinica plant regenerated fromthe tissue culture of claim 6, said plant having all of thecharacteristics of Brassica rapa var. nipposinica ORIGAMI, wherein arepresentative sample of seed of said Brassica rapa var. nipposinicahaving been deposited under NCIMB No ______.
 8. A Brassica rapa var.nipposinica leaf produced from the plant of claim
 2. 9. A method forproducing a Brassica rapa var. nipposinica leaf comprising a) growingthe Brassica rapa var. nipposinica plant of claim 2 to produce aBrassica rapa var. nipposinica leaf, and b) harvesting said Brassicarapa var. nipposinica leaf.
 10. A Brassica rapa var. nipposinica leafproduced by the method of claim
 9. 11. A method for producing a Brassicarapa var. nipposinica seed comprising crossing a first parent Brassicarapa var. nipposinica plant with a second parent Brassica rapa var.nipposinica plant and harvesting the resultant Brassica rapa var.nipposinica seed, wherein said first parent Brassica rapa var.nipposinica plant and/or the second parent Brassica rapa var.nipposinica plant is the Brassica rapa var. nipposinica plant of claim2.
 12. An F1 Brassica rapa var. nipposinica seed produced by the methodof claim
 11. 13. A method for producing a Brassica rapa var. nipposinicaseed comprising self-pollinating the Brassica rapa var. nipposinicaplant of claim 2 and harvesting the resultant Brassica rapa var.nipposinica seed.
 14. A Brassica rapa var. nipposinica seed produced bythe method of claim
 13. 15. A method of producing a Brassica rapa var.nipposinica plant derived from the Brassica rapa var. nipposinicaORIGAMI, the method comprising (a) crossing the plant of claim 2 with asecond Brassica rapa var. nipposinica plant to produce a progeny plant.16. The method of claim 15 further comprising the step of: (b) crossingthe progeny plant derived from Brassica rapa var. nipposinica ORIGAMIwith itself or a second plant to produce a seed of progeny plant ofsubsequent generation; (c) growing the progeny plant of the subsequentgeneration from the seed (d) crossing the progeny plant of thesubsequent generation with itself or the second plant, to produce aBrassica rapa var. nipposinica plant derived from the Brassica rapa var.nipposinica ORIGAMI.
 17. The method of claim 16 further comprising thestep of: (e) repeating steps (b) and/or (c) to produce a Brassica rapavar. nipposinica plant derived from the Brassica rapa var. nipposinicaORIGAMI.
 18. The plant of claim 2 comprising at least one single locusconversion and otherwise essentially all of the characteristics ofORIGAMI listed in Table 1 when grown under the same environmentalconditions.
 19. The plant of claim 18 wherein the at least one singlelocus conversion confers said plant with herbicide resistance.
 20. Theplant of claim 18 wherein the at least one single locus conversion is anartificially mutated gene or nucleotide sequence.
 21. A method ofintroducing a desired trait into Brassica rapa var. nipposinica ORIGAMIcomprising: (a) crossing a Brassica rapa var. nipposinica ORIGAMI plantgrown from Brassica rapa var. nipposinica ORIGAMI seed, wherein arepresentative sample of seed has been deposited under NCIMB No. ______,with another lettuce plant that comprises a desired trait to produce F1progeny plants; (b) selecting one or more progeny plants that have thedesired trait to produce selected progeny plants; (c) crossing theselected progeny plants with the Brassica rapa var. nipposinica ORIGAMIplants to produce backcross progeny plants; (d) selecting for backcrossprogeny plants that have the desired trait and all of the physiologicaland morphological characteristics of Brassica rapa var. nipposinicaORIGAMI listed in Table 1 when grown in the same environmentalconditions to produce selected backcross progeny plants; and (e)repeating steps (c) and (d) three or more times in succession to produceselected fourth or higher backcross progeny plants that comprise thedesired trait and all of the physiological and morphologicalcharacteristics of Brassica rapa var. nipposinica ORIGAMI listed inTable 1 when grown in the same environmental conditions.